HHS Public Access Author manuscript Author Manuscript
J Pediatr. Author manuscript; available in PMC 2017 July 01. Published in final edited form as: J Pediatr. 2016 July ; 174: 45–51.e5. doi:10.1016/j.jpeds.2016.03.058.
Blood Cytokine Profiles Associated with Distinct Patterns of Bronchopulmonary Dysplasia among Extremely Low Birth Weight Infants
Author Manuscript
Carl T. D’Angio, MD1, Namasivayam Ambalavanan, MD2, Waldemar A. Carlo, MD2, Scott A. McDonald, BS3, Kristin Skogstrand, PhD4, David M. Hougaard, MD, DSc4, Seetha Shankaran, MD5, Ronald N. Goldberg, MD6, Richard A. Ehrenkranz, MD7, Jon E. Tyson, MD, MPH8, Barbara J. Stoll, MD9, Abhik Das, PhD10, and Rosemary D. Higgins, MD11 on behalf of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network* 1Strong
Children’s Research Center, University of Rochester School of Medicine and Dentistry, Rochester, NY 2Department 3Statistics
of Pediatrics, University of Alabama at Birmingham, Birmingham, AL
and Epidemiology Unit, RTI International, Research Triangle Park, NC
4Danish
Centre for Neonatal Screening, Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark
Author Manuscript
5Department
of Pediatrics, Wayne State University, Detroit, MI
6Department
of Pediatrics, Duke University, Durham, NC
7Department
of Pediatrics, Yale University School of Medicine, New Haven, CT
8Department
of Pediatrics, University of Texas Medical School at Houston, Houston, TX
9Emory
University School of Medicine, Department of Pediatrics, Children’s Healthcare of Atlanta, Atlanta, GA
10Statistics
and Epidemiology Unit, RTI International, Rockville, MD
11Eunice
Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
Author Manuscript
Abstract Objective—To explore differences in blood cytokine profiles among distinct BPD patterns. Study design—We evaluated blood spots collected from 943 infants born at ≤1000 grams and surviving to 28 days on postnatal days 1, 3, 7, 14, and 21 for 25 cytokines. Infants were assigned
Corresponding Author: Carl T. D’Angio, MD, Box 651, Neonatology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, Phone: (585) 273-4911, Fax: (585) 461-3614, [email protected]. *List of additional members of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Neonatal Research Network is available at www.jpeds.com (Appendix). The authors declare no conflicts of interest.
D’Angio et al.
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Author Manuscript
to the following lung disease patterns: (1) no lung disease (NLD), (2) respiratory distress syndrome (RDS) without BPD, (3) classic BPD (persistent exposure to supplemental oxygen until 28 days of age) or (4) atypical BPD (period without supplemental oxygen before 28 days). Median cytokine levels for infants with BPD were compared with the interquartile range (IQR) of results among infants with NLD. Results—The distribution of enrolled infants by group was as follows: 69 (NLD), 73 (RDS), 381 (classic BPD), and 160 (atypical BPD). The remaining 260 infants could not be classified due to missing data (104) or not fitting a pre-defined pattern (156). Median levels of 3 cytokines (elevated IL-8, MMP-9; decreased GM-CSF) fell outside the IQR for at least 2 time points in both atypical and classic BPD infants. Profiles of 7 cytokines (IL-6, -10, -18, MIP-1α, CRP, BDNF, RANTES) differed between infants with classic and atypical BPD.
Author Manuscript
Conclusion—Blood cytokine profiles may differ between infants developing classic and atypical BPD. These dissimilarities suggest the possibility that differing mechanisms could explain the varied patterns of pathophysiology of lung disease in extremely premature infants. Keywords Premature infant; respiratory distress syndrome; chronic lung disease; inflammatory markers Many investigators have described the association of altered lung fluid cytokine levels with the development of bronchopulmonary dysplasia (BPD), but these studies are limited by their small size and their inability to obtain samples from non-intubated infants [1–8]. Fewer studies have explored the potential association between variations in blood cytokine levels and BPD [9–12].
Author Manuscript
Some authors have described a “new” BPD associated with improved survival in smaller infants and decreased incidence of the more classic, severe form of BPD due to the availability of newer management approaches [13, 14]. We have previously described four patterns of respiratory course among infants ≤ 1250 grams birth weight, based on oxygen administration: (1) normal – no clinical or radiological evidence of lung disease; (2) respiratory distress syndrome (RDS) – typical RDS, with no supplemental oxygen administration at 28 days of age; (3) classic BPD – acute lung disease that does not resolve, leading to oxygen administration and radiologic evidence of BPD at 28 days; and (4) atypical BPD – initial resolution of RDS, if present, followed by subsequent development of oxygen requirement that persists through 28 days [13]. Although it would not be appropriate to conflate “new” BPD with the atypical group we describe, these infants may represent a unique group in which to study the pathogenesis of the “new” BPD.
Author Manuscript
The Eunice Kennedy Shriver National Institutes of Child Health and Human Development’s Neonatal Research Network (NRN) has performed cytokine assays on blood spots from over 1000 premature infants. We have previously reported that adding cytokine results to clinical predictors improved the ability of a multivariate model to predict the later development of BPD [11]. However, the modeling did not take into account potential subtypes of BPD. We re-analyzed the cytokine data, specifically dividing subjects into groups with classic and atypical BPD. We hypothesized that the patterns of expression of a limited number of blood
J Pediatr. Author manuscript; available in PMC 2017 July 01.
D’Angio et al.
Page 3
Author Manuscript
cytokines would differ between infants with classic BPD, infants with atypical BPD, and infants without BPD. We speculated that these differences in cytokine levels could serve as biomarkers of disease type and form the focus of further, hypothesis-driven exploration of the pathogenesis of BPD.
METHODS
Author Manuscript Author Manuscript
This study analyzed data from a prospective cohort study of blood cytokine levels among premature infants that has been reported previously [11, 15]. Briefly, whole blood spots were collected on filter paper from infants with birth weights between 401 and 1000 grams born at one of 17 NRN centers between 1999 and 2002. Samples were collected within 4 hours after birth (“day 1”) and then between 2–4 days (“day 3”), 6–8 days (“day 7”), 11–17 days (“day 14”) and 18–24 days (“day 21”). Samples were dried using a desiccator, frozen immediately and analyzed later for levels of 25 cytokines using a multiplex, bead-based luminescent immunoassay (Luminex®, Austin, TX), as previously described [16]. The cytokine levels measured included interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-10, IL-12, IL-17, IL-18, tumor necrosis factor (TNF)-α, TNF-β, RANTES, brain-derived neurotrophic factor (BDNF), C-reactive protein (CRP), granulocyte/ macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, macrophage chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, matrix metalloproteinase (MMP)-9, neurotrophin (NT) 4, soluble IL-6 receptor (SIR), transforming growth factor (TGF)-β, and triggering receptor expressed on myeloid cells 1 (TREM1). Clinical data, including inspired concentration of oxygen (FiO2) on days 1, 3, 7, 14, 21 and 28 and at 36 weeks’ postmenstrual age (PMA), were collected from the medical record. The original study was approved by each center’s Institutional Review Board, and parents gave informed permission for the study. This analysis used only de-identified data collected at the time of the original study.
Author Manuscript
Infants were divided into groups to correspond with our previously reported patterns of lung disease [13], according to oxygen administration at 1, 4, 7, 14, 21 and 28 days of age. The means of oxygen administration was not included in the definition. A group with “no lung disease” (NLD) was defined by having FiO2 < 0.25 at days 1, 4, 7, 14, 21 and 28 days. A group was identified as having “respiratory distress syndrome without BPD” (RDS) by having FiO2 ≥ 0.25 within the first 21 days, FiO2 < 0.25 at day 3 and/or day 7 and FiO2 0.21), 70 (44%) had mild BPD (FiO2 0.21), and 47 (29%) had missing data for FiO2 at 36 weeks’ PMA (and were not included in the 36 week data). Of infants with classic BPD assessed at 36 weeks’ PMA, 25 (7%) had died, 125 (33%) had moderate/severe BPD, 80 (21%) had mild BPD, and 151 (40%) had missing data for FiO2 at 36 weeks’ PMA (and were not included in the 36 week data). Baseline and clinical variables of the groups, including of the group of infants unable to be categorized, are described in the Table. As expected, there were several differences in baseline and clinical variables among the disease groups and between the classifiable and non-classifiable infants. Of the 260 infants who could not be classified, 156 were indeterminate (≥1 FiO2 measurement 1 missing FiO2 value).
Author Manuscript
As expected from the definitions, infants with BPD of either type had higher FiO2 at 28 days (Figure 2), with mean FiO2 in the NLD and RDS groups of 0.21. Infants with atypical BPD had FiO2 at days 1 and 3 more similar to the RDS group than to the classic BPD group. Thereafter, the atypical BPD group had FiO2 values intermediate between those of the classic BPD group and the other groups. Also as expected from the group definitions, nonclassified infants’ FiO2 values fell between those of the BPD groups (Figure 3; available at www.jpeds.com). No median cytokine levels in the RDS group fell outside the interquartile range (IQR) of infants in the NLD group (Figure 4). To simplify presentation, Figure 4 depicts only values for infants in the moderate-to-severe BPD groups, except in the case of GM-CSF. For the majority of cytokines, median cytokine levels in neither the atypical nor the classic BPD groups (whether all BPD or only moderate-to-severe BPD) fell outside the IQR of the NLD
J Pediatr. Author manuscript; available in PMC 2017 July 01.
D’Angio et al.
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Author Manuscript Author Manuscript
group. However, median levels of IL-8 and MMP-9 fell above the IQR of the NLD infants for at least two determinations in both the atypical BPD and classic BPD groups. Median levels of CRP, IL-6, IL-10 and MIP-1α had at least two determinations above the IQR of the NLD infants in the classic BPD group, and median levels of IL-18 were above the IQR of the NLD group at least twice in the atypical BPD groups. In addition, GM-CSF median levels were below the IQR of the NLD group at least twice in both the atypical and classic BPD groups. Median BDNF and RANTES levels were below the IQR of the NLD group at least twice in the classic BPD group. In all cases, except that of GM-CSF, cytokine concentrations among the groups with moderate- to-severe BPD were more extreme than values among the BPD group as a whole and accounted for the two values outside the IQR of the NLD group (data not shown). None of the other analytes displayed two values outside the IQR of the NLD infants (Figure 5; available at www.jpeds.com). Sensitivity analyses using FiO2 0.21 (rather than
J Pediatr. Author manuscript; available in PMC 2017 July 01. Published in final edited form as: J Pediatr. 2016 July ; 174: 45–51.e5. doi:10.1016/j.jpeds.2016.03.058.
Blood Cytokine Profiles Associated with Distinct Patterns of Bronchopulmonary Dysplasia among Extremely Low Birth Weight Infants
Author Manuscript
Carl T. D’Angio, MD1, Namasivayam Ambalavanan, MD2, Waldemar A. Carlo, MD2, Scott A. McDonald, BS3, Kristin Skogstrand, PhD4, David M. Hougaard, MD, DSc4, Seetha Shankaran, MD5, Ronald N. Goldberg, MD6, Richard A. Ehrenkranz, MD7, Jon E. Tyson, MD, MPH8, Barbara J. Stoll, MD9, Abhik Das, PhD10, and Rosemary D. Higgins, MD11 on behalf of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network* 1Strong
Children’s Research Center, University of Rochester School of Medicine and Dentistry, Rochester, NY 2Department 3Statistics
of Pediatrics, University of Alabama at Birmingham, Birmingham, AL
and Epidemiology Unit, RTI International, Research Triangle Park, NC
4Danish
Centre for Neonatal Screening, Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark
Author Manuscript
5Department
of Pediatrics, Wayne State University, Detroit, MI
6Department
of Pediatrics, Duke University, Durham, NC
7Department
of Pediatrics, Yale University School of Medicine, New Haven, CT
8Department
of Pediatrics, University of Texas Medical School at Houston, Houston, TX
9Emory
University School of Medicine, Department of Pediatrics, Children’s Healthcare of Atlanta, Atlanta, GA
10Statistics
and Epidemiology Unit, RTI International, Rockville, MD
11Eunice
Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
Author Manuscript
Abstract Objective—To explore differences in blood cytokine profiles among distinct BPD patterns. Study design—We evaluated blood spots collected from 943 infants born at ≤1000 grams and surviving to 28 days on postnatal days 1, 3, 7, 14, and 21 for 25 cytokines. Infants were assigned
Corresponding Author: Carl T. D’Angio, MD, Box 651, Neonatology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, Phone: (585) 273-4911, Fax: (585) 461-3614, [email protected]. *List of additional members of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Neonatal Research Network is available at www.jpeds.com (Appendix). The authors declare no conflicts of interest.
D’Angio et al.
Page 2
Author Manuscript
to the following lung disease patterns: (1) no lung disease (NLD), (2) respiratory distress syndrome (RDS) without BPD, (3) classic BPD (persistent exposure to supplemental oxygen until 28 days of age) or (4) atypical BPD (period without supplemental oxygen before 28 days). Median cytokine levels for infants with BPD were compared with the interquartile range (IQR) of results among infants with NLD. Results—The distribution of enrolled infants by group was as follows: 69 (NLD), 73 (RDS), 381 (classic BPD), and 160 (atypical BPD). The remaining 260 infants could not be classified due to missing data (104) or not fitting a pre-defined pattern (156). Median levels of 3 cytokines (elevated IL-8, MMP-9; decreased GM-CSF) fell outside the IQR for at least 2 time points in both atypical and classic BPD infants. Profiles of 7 cytokines (IL-6, -10, -18, MIP-1α, CRP, BDNF, RANTES) differed between infants with classic and atypical BPD.
Author Manuscript
Conclusion—Blood cytokine profiles may differ between infants developing classic and atypical BPD. These dissimilarities suggest the possibility that differing mechanisms could explain the varied patterns of pathophysiology of lung disease in extremely premature infants. Keywords Premature infant; respiratory distress syndrome; chronic lung disease; inflammatory markers Many investigators have described the association of altered lung fluid cytokine levels with the development of bronchopulmonary dysplasia (BPD), but these studies are limited by their small size and their inability to obtain samples from non-intubated infants [1–8]. Fewer studies have explored the potential association between variations in blood cytokine levels and BPD [9–12].
Author Manuscript
Some authors have described a “new” BPD associated with improved survival in smaller infants and decreased incidence of the more classic, severe form of BPD due to the availability of newer management approaches [13, 14]. We have previously described four patterns of respiratory course among infants ≤ 1250 grams birth weight, based on oxygen administration: (1) normal – no clinical or radiological evidence of lung disease; (2) respiratory distress syndrome (RDS) – typical RDS, with no supplemental oxygen administration at 28 days of age; (3) classic BPD – acute lung disease that does not resolve, leading to oxygen administration and radiologic evidence of BPD at 28 days; and (4) atypical BPD – initial resolution of RDS, if present, followed by subsequent development of oxygen requirement that persists through 28 days [13]. Although it would not be appropriate to conflate “new” BPD with the atypical group we describe, these infants may represent a unique group in which to study the pathogenesis of the “new” BPD.
Author Manuscript
The Eunice Kennedy Shriver National Institutes of Child Health and Human Development’s Neonatal Research Network (NRN) has performed cytokine assays on blood spots from over 1000 premature infants. We have previously reported that adding cytokine results to clinical predictors improved the ability of a multivariate model to predict the later development of BPD [11]. However, the modeling did not take into account potential subtypes of BPD. We re-analyzed the cytokine data, specifically dividing subjects into groups with classic and atypical BPD. We hypothesized that the patterns of expression of a limited number of blood
J Pediatr. Author manuscript; available in PMC 2017 July 01.
D’Angio et al.
Page 3
Author Manuscript
cytokines would differ between infants with classic BPD, infants with atypical BPD, and infants without BPD. We speculated that these differences in cytokine levels could serve as biomarkers of disease type and form the focus of further, hypothesis-driven exploration of the pathogenesis of BPD.
METHODS
Author Manuscript Author Manuscript
This study analyzed data from a prospective cohort study of blood cytokine levels among premature infants that has been reported previously [11, 15]. Briefly, whole blood spots were collected on filter paper from infants with birth weights between 401 and 1000 grams born at one of 17 NRN centers between 1999 and 2002. Samples were collected within 4 hours after birth (“day 1”) and then between 2–4 days (“day 3”), 6–8 days (“day 7”), 11–17 days (“day 14”) and 18–24 days (“day 21”). Samples were dried using a desiccator, frozen immediately and analyzed later for levels of 25 cytokines using a multiplex, bead-based luminescent immunoassay (Luminex®, Austin, TX), as previously described [16]. The cytokine levels measured included interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-10, IL-12, IL-17, IL-18, tumor necrosis factor (TNF)-α, TNF-β, RANTES, brain-derived neurotrophic factor (BDNF), C-reactive protein (CRP), granulocyte/ macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, macrophage chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, matrix metalloproteinase (MMP)-9, neurotrophin (NT) 4, soluble IL-6 receptor (SIR), transforming growth factor (TGF)-β, and triggering receptor expressed on myeloid cells 1 (TREM1). Clinical data, including inspired concentration of oxygen (FiO2) on days 1, 3, 7, 14, 21 and 28 and at 36 weeks’ postmenstrual age (PMA), were collected from the medical record. The original study was approved by each center’s Institutional Review Board, and parents gave informed permission for the study. This analysis used only de-identified data collected at the time of the original study.
Author Manuscript
Infants were divided into groups to correspond with our previously reported patterns of lung disease [13], according to oxygen administration at 1, 4, 7, 14, 21 and 28 days of age. The means of oxygen administration was not included in the definition. A group with “no lung disease” (NLD) was defined by having FiO2 < 0.25 at days 1, 4, 7, 14, 21 and 28 days. A group was identified as having “respiratory distress syndrome without BPD” (RDS) by having FiO2 ≥ 0.25 within the first 21 days, FiO2 < 0.25 at day 3 and/or day 7 and FiO2 0.21), 70 (44%) had mild BPD (FiO2 0.21), and 47 (29%) had missing data for FiO2 at 36 weeks’ PMA (and were not included in the 36 week data). Of infants with classic BPD assessed at 36 weeks’ PMA, 25 (7%) had died, 125 (33%) had moderate/severe BPD, 80 (21%) had mild BPD, and 151 (40%) had missing data for FiO2 at 36 weeks’ PMA (and were not included in the 36 week data). Baseline and clinical variables of the groups, including of the group of infants unable to be categorized, are described in the Table. As expected, there were several differences in baseline and clinical variables among the disease groups and between the classifiable and non-classifiable infants. Of the 260 infants who could not be classified, 156 were indeterminate (≥1 FiO2 measurement 1 missing FiO2 value).
Author Manuscript
As expected from the definitions, infants with BPD of either type had higher FiO2 at 28 days (Figure 2), with mean FiO2 in the NLD and RDS groups of 0.21. Infants with atypical BPD had FiO2 at days 1 and 3 more similar to the RDS group than to the classic BPD group. Thereafter, the atypical BPD group had FiO2 values intermediate between those of the classic BPD group and the other groups. Also as expected from the group definitions, nonclassified infants’ FiO2 values fell between those of the BPD groups (Figure 3; available at www.jpeds.com). No median cytokine levels in the RDS group fell outside the interquartile range (IQR) of infants in the NLD group (Figure 4). To simplify presentation, Figure 4 depicts only values for infants in the moderate-to-severe BPD groups, except in the case of GM-CSF. For the majority of cytokines, median cytokine levels in neither the atypical nor the classic BPD groups (whether all BPD or only moderate-to-severe BPD) fell outside the IQR of the NLD
J Pediatr. Author manuscript; available in PMC 2017 July 01.
D’Angio et al.
Page 5
Author Manuscript Author Manuscript
group. However, median levels of IL-8 and MMP-9 fell above the IQR of the NLD infants for at least two determinations in both the atypical BPD and classic BPD groups. Median levels of CRP, IL-6, IL-10 and MIP-1α had at least two determinations above the IQR of the NLD infants in the classic BPD group, and median levels of IL-18 were above the IQR of the NLD group at least twice in the atypical BPD groups. In addition, GM-CSF median levels were below the IQR of the NLD group at least twice in both the atypical and classic BPD groups. Median BDNF and RANTES levels were below the IQR of the NLD group at least twice in the classic BPD group. In all cases, except that of GM-CSF, cytokine concentrations among the groups with moderate- to-severe BPD were more extreme than values among the BPD group as a whole and accounted for the two values outside the IQR of the NLD group (data not shown). None of the other analytes displayed two values outside the IQR of the NLD infants (Figure 5; available at www.jpeds.com). Sensitivity analyses using FiO2 0.21 (rather than
